Ulla Vanleeuw

Plasma MMP1 and MMP8 expression in breast cancer: protective role of MMP8 against lymph node metastasis.

BackgroundElevated levels of matrix metalloproteinases have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer.MethodsA total of 208 breast cancer patients, of which 21 with inflammatory breast cancer, and 42 healthy controls were included. Plasma MMP1, MMP8 and MMP13 levels were measured using ELISA and correlated with clinicopathological characteristics.ResultsMedian plasma MMP1 levels were higher in controls than in breast cancer patients (3.45 vs. 2.01 ng/ml), while no difference was found for MMP8 (10.74 vs. 10.49 ng/ml). ROC analysis for MMP1 revealed an AUC of 0.67, sensitivity of 80% and specificity of 24% at a cut-off value of 4.24 ng/ml. Plasma MMP13 expression could not be detected. No correlation was found between MMP1 and MMP8 levels. We found a trend of lower MMP1 levels with increasing tumour size (p = 0.07); and higher MMP8 levels with premenopausal status (p = 0.06) and NPI (p = 0.04). The median plasma MMP1 (p = 0.02) and MMP8 (p = 0.007) levels in the non-inflammatory breast cancer patients were almost twice as high as those found in the inflammatory breast cancer patients. Intriguingly, plasma MMP8 levels were positively associated with lymph node involvement but showed a negative correlation with the risk of distant metastasis. Both controls and lymph node negative patients (pN0) had lower MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (p = 0.001); and showed a trend for higher MMP8 levels compared to patients with extensive lymph node involvement (pN3) and a strong predisposition to distant metastasis (p = 0.11). Based on the hypothesis that blood and tissue protein levels are in reverse association, these results suggest that MMP8 in the tumour may have a protective effect against lymph node metastasis.ConclusionIn summary, we observed differences in MMP1 and MMP8 plasma levels between healthy controls and breast cancer patients as well as between breast cancer patients. Interestingly, our results suggest that MMP8 may affect the metastatic behaviour of breast cancer cells through protection against lymph node metastasis, underlining the importance of anti-target identification in drug development.

The Heat Shock Protein 90 Inhibitor IPI-504 Induces KIT Degradation, Tumor Shrinkage, and Cell Proliferation Arrest in Xenograft Models of Gastrointestinal Stromal Tumors

The activity of the receptor tyrosine kinase KIT is crucial for gastrointestinal stromal tumor (GIST) growth and survival. Imatinib and sunitinib are very effective in advanced GIST, but have no curative potential. The observation that heat shock protein 90 (HSP90) inhibition results in KIT degradation prompted us to assess the efficacy of the HSP90 inhibitor retaspimycin hydrochloride (IPI-504) alone or in combination with imatinib or sunitinib in two GIST xenografts with distinctive KIT mutations. Nude mice were grafted with human GIST carrying KIT exon 13 (GIST-882; n = 59) or exon 11 (GIST-PSW; n = 44) mutations and dosed with imatinib (50 mg/kg twice daily), sunitinib (40 mg/kg once daily), IPI-504 (100 mg/kg 3 times per week), IPI-504 + imatinib, or IPI-504 + sunitinib. We evaluated tumor volume, proliferation and apoptosis, KIT expression and activation, as well as adverse events during treatment. Treatment with IPI-504 alone resulted in tumor regression, proliferation arrest, and induction of tumor necrosis. We documented downregulation of KIT and its signaling cascade in IPI-504–treated animals. Treatment effects were enhanced by combining IPI-504 with imatinib or sunitinib. On histologic examination, liver damage was frequently observed in animals exposed to combination treatments. In conclusion, IPI-504 shows consistent antitumor activity and induces KIT downregulation in GIST, as a single agent, and is more potent in combination with imatinib or sunitinib. The sequence of drug administration in the combination arms warrants further studies. Mol Cancer Ther; 10(10); 1897–908. ©2011 AACR.

Phosphoinositide 3-Kinase Inhibitors Combined with Imatinib in Patient-Derived Xenograft Models of Gastrointestinal Stromal Tumors: Rationale and Efficacy

Introduction: The PI3K signaling pathway drives tumor cell proliferation and survival in gastrointestinal stromal tumor (GIST). We tested the in vivo efficacy of three PI3K inhibitors (PI3Ki) in patient-derived GIST xenograft models. Experimental Design: One hundred and sixty-eight nude mice were grafted with human GIST carrying diverse KIT genotypes and PTEN genomic status. Animals were dosed orally for two weeks as follows: control group (untreated); imatinib (IMA); PI3Ki (BKM120—buparlisib, BEZ235, or BYL719) or combinations of imatinib with a PI3Ki. Western blotting, histopathology, and tumor volume evolution were used for the assessment of treatment efficacy. Furthermore, tumor regrowth was evaluated for three weeks after treatment cessation. Results: PI3Ki monotherapy showed a significant antitumor effect, reflected in tumor volume reduction or stabilization, inhibitory effects on mitotic activity, and PI3K signaling inhibition. The IMA+PI3Ki combination remarkably improved the efficacy of either single-agent treatment with more pronounced tumor volume reduction and enhanced proapoptotic effects over either single agent. Response to IMA+PI3Ki was found to depend on the KIT genotype and specific model-related molecular characteristics. Conclusion: IMA+PI3Ki has significant antitumor efficacy in GIST xenografts as compared with single-agent treatment, resulting in more prominent tumor volume reduction and enhanced induction of apoptosis. Categorization of GIST based on KIT genotype and PI3K/PTEN genomic status combined with dose optimization is suggested for patient selection for clinical trials exploring such combinations. Clin Cancer Res; 20(23); 6071–82. ©2014 AACR.

Pazopanib, a Receptor Tyrosine Kinase Inhibitor, Suppresses Tumor Growth through Angiogenesis in Dedifferentiated Liposarcoma Xenograft Models

INTRODUCTION: The rarity of dedifferentiated liposarcoma (DDLPS) and the lack of experimental DDLPS models limit the development of novel therapeutic strategies. Pazopanib (PAZ) is a tyrosine kinase inhibitor that is approved for the treatment of non-adipocytic advanced soft tissue sarcoma. The activity of this agent has not yet been properly explored in preclinical liposarcoma models nor in a randomized phase Ш clinical trial in this entity. The aim of the present study was to investigate whether PAZ had antitumor activity in DDLPS models in vivo. MATERIAL AND METHODS: We established two patient-derived DDLPS xenograft models (UZLX-STS3 and UZLX-STS5) through implantation of tumor material from sarcoma patients in athymic nude NMRI mice. An animal model of the SW872 liposarcoma cell line was also used. To investigate the efficacy of PAZ in vivo, mice bearing tumors were treated for 2 weeks with sterile water, doxorubicin (1.2 mg/kg, intraperitoneally, twice per week), PAZ [40 mg/kg, orally (p.o.), twice per day], or PAZ plus doxorubicin (same schedules as for single treatments). RESULTS: Patient-derived xenografts retained the histologic and molecular features of DDLPS. PAZ significantly delayed tumor growth by decreasing proliferation and inhibited angiogenesis in all models tested. Combining the angiogenesis inhibitor with an anthracycline did not show superior efficacy. CONCLUSION: These results suggest that PAZ has potential antitumor activity in DDLPS primarily through antiangiogenic effects and therefore should be explored in clinical trials.

Plasma MMP1, MMP8 and MMP13 expression in breast cancer: protective role of MMP8 against lymph node metastasis

Elevated levels of matrix metalloproteinases (MMPs) have been found to associate with poor prognosis in various carcinomas. This study aimed at evaluating plasma levels of the collagenases MMP1, MMP8 and MMP13 as diagnostic and prognostic markers of breast cancer. Using ELISA, plasma levels of MMP1, MMP8 and MMP13 were measured in 42 control individuals and in 208 patients – of which 21 were inflammatory breast cancer patients – and were correlated with standard clinicopathological data. Plasma MMP1 levels were higher in breast cancer patients than in control individuals, while the opposite was true for MMP8. Plasma MMP13 levels could not be detected. We found a negative correlation of plasma MMP1 with tumour size (P = 0.07); and a positive association of MMP8 with the premenopausal status (P = 0.06), Nottingham Prognostic Index (P = 0.06) and Her2 expression (P = 0.07). Further, a twofold decrease in MMP1 (P = 0.025) and MMP8 (P = 0.007) levels was observed in inflammatory breast cancer patients, a very rare and not well understood aggressive disease. Most interestingly, we observed a peculiar relation between plasma MMP8 levels and lymph node metastasis. We found that both control individuals and patients without lymph node involvement (pN0) have lower plasma MMP8 levels than patients with moderate lymph node involvement (pN1, pN2) (P = 0.001); and that they show a trend for higher MMP8 levels as compared with patients with extensive lymph node metastasis (pN3) and a strong predisposition to distant metastasis. In summary, we observed differences in MMP1 and MMP8 plasma levels between distinct breast cancer patient groups. As it is not clear to date whether MMPs in blood and body fluids have a physiological function per se, we hypothesize that altered levels in blood reflect local changes in the extracellular microenvironment. As such, a positive association of blood MMP levels with clinical characteristics and tumour features reflects a negative association with tissue MMP levels and vice versa. Therefore, our results suggest that both MMP1 and MMP8 in the tumour may contribute to the aggressive phenotype of inflammatory breast carcinomas. Interestingly, our results suggest that tumour MMP8 expression may affect the metastatic behaviour of breast cancer cells with a greater protective effect against lymph node metastasis than against distant metastasis.

Cabozantinib is active against human gastrointestinal stromal tumor xenografts carrying different KIT mutations

In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845–52. ©2016 AACR.

Therapeutic Efficacy Assessment of CK6, a Monoclonal KIT Antibody, in a Panel of Gastrointestinal Stromal Tumor Xenograft Models

We evaluated the efficacy of CK6, a KIT monoclonal antibody, in a panel of human gastrointestinal stromal tumor (GIST) xenograft models. Nude mice were bilaterally transplanted with human GIST xenografts (four patient derived and two cell line derived), treated for 3 weeks, and grouped as follows: control (untreated); CK6 (40 mg/kg, 3 × weekly); imatinib (50 mg/kg, twice daily); sunitinib (40 mg/kg, once daily); imatinib + CK6; sunitinib + CK6 (same doses and schedules as in the single-agent treatments). Tumor volume assessment, Western blot analysis, and histopathology were used for evaluation of efficacy. Statistical analysis was performed using Mann-Whitney U (MWU) and Wilcoxon matched-pairs tests. CK6 as a single agent only reduced tumor growth rate in the UZLX-GIST3 model (P = .053, MWU compared to control), while in none of the other GIST models an effect on tumor growth rate was observed. CK6 did not result in significant anti-proliferative or pro-apoptotic effects in any of the GIST models, and moreover, CK6 did not induce a remarkable inhibition of KIT activation. Furthermore, no synergistic effect of combining CK6 with tyrosine kinase inhibitors (TKIs) was observed. Conversely, in certain GIST xenografts, anti-tumor effects seemed to be inferior under combination treatment compared to single-agent TKI treatment. In the GIST xenografts tested, the anti-tumor efficacy of CK6 was limited. No synergy was observed on combination of CK6 with TKIs in these GIST models. Our findings highlight the importance of using relevant in vivo human tumor xenograft models in the preclinical assessment of drug combination strategies.

Abstract 1460: Establishment and characterization of a panel of patient-derived soft tissue sarcoma (STS) xenograft models for in vivo testing of novel therapeutic approaches

Background: STS constitutes a rare and very heterogeneous family of tumors of mesenchymal origin. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts was established and is currently expanded in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumor specimens from consenting patients with STS, treated at the University Hospitals Leuven, Belgium. We mainly focused on STS subtypes which harbor specific genetic alterations (e.g. translocations, gene amplifications). Once tumor growth was observed, pieces of tissue were re-transplanted to the next generation of mice. At each passage tumor fragments were collected for histological and molecular characterization. Fluorescence in situ hybridization (FISH) was performed to confirm specific genomic alterations in selected xenografts. A STS xenograft model was considered established after observing maintained histological and molecular features for at least two passages. Results: Until now a total of 78 STS patient samples have been transplanted. Fifteen well-characterized, stable STS models have already been established, maintaining the features of the original tumor: myxofibrosarcoma (4 models), dedifferentiated liposarcoma (2), synovial sarcoma (2), epithelioid haemangioendothelioma (1), malignant peripheral nerve sheath tumor (2), mesenchymal chondrosarcoma (1), leiomyosarcoma (2) and sarcoma not otherwise specified (1). Some of these models have already been successfully used for in vivo testing of novel agents, including tyrosine kinase inhibitors or cytotoxic (pro-)drugs. Twelve other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of “established model”. Conclusion: This newly established, representative panel of STS xenografts is characterized by stable histological and molecular features reflecting the pheno- and genotype of the original patient samples. This panel is thus well suited for in vivo drug testing of innovative agents. The unique availability of models of some ultra-rare entities will also allow us to study the biology of these diseases. The platform is available for collaboration with academic and commercial partners (contact patrick.schoffski@uzleuven.be). Citation Format: Haifu Li, Jasmien Cornillie, Agnieszka Wozniak, Thomas Van Looy, Yemarshet Gebreyohannes, Jasmien Wellens, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Establishment and characterization of a panel of patient-derived soft tissue sarcoma (STS) xenograft models for in vivo testing of novel therapeutic approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1460. doi:10.1158/1538-7445.AM2015-1460

In vivo antitumoral efficacy of PhAc-ALGP-doxorubicin, an enzyme-activated doxorubicin prodrug, in patient-derived soft tissue sarcoma xenograft models

Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo, or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology, and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of 2 weeks after treatment cessation. Although tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared with control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared with doxo in all patient-derived xenograft models tested. Administration of a 30- to 40-fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. Mol Cancer Ther; 16(8); 1566–75. ©2017 AACR.

Abstract 775: Anti-tumor effects of dovitinib, a multi-target kinase inhibitor, in patient-derived gastrointestinal stromal tumor (GIST) xenograft models

Background: The majority of GISTs are driven by KIT/PDGFRA gain of function mutations. Advanced GIST is routinely treated with tyrosine kinase inhibitors (TKI; e.g imatinib, sunitinib and regorafenib). However, with time, heterogeneous resistance to these agents occurs, mainly due to acquired mutations in KIT. We tested the efficacy of dovitinib, which acts against VEGFR, FGFR, FLT3, PDGFRB and KIT, using patient-derived GIST xenograft models. Methods: NMRI nu/nu mice (n = 47) were bilaterally transplanted with the human GIST xenografts: a dose-dependent-imatinib-sensitive model UZLX-GIST2 (KIT: p.A502_Y503dup) or a model resistant to both imatinib and sunitinib, UZLX-GIST9 (KIT: p.P577del+p.W557LfsX5+p.D820G). Mice were divided to four treatment groups: control (untreated), imatinib (50 mg/kg/bid p.o.), imatinib (100 mg/kg/bid p.o.) and dovitinib (30 mg/kg/qd p.o.). Efficacy was assessed by tumor volume measurement (3x/week), histopathology [hematoxylin & eosin staining (HE p Conclusion: The multi-kinase inhibitor dovitinib showed anti-tumor efficacy in GIST xenograft models, though the effect was more pronounced in KIT exon 9 mutant (UZLX-GIST2). The decrease in MVD in both models suggested that the anti-tumor effect in these models was at least partially mediated by an anti-angiogenic effect of dovitinib. 1 Antonescu et al. Clin Cancer Res. 2005; 11:4182-90 Citation Format: Yemarshet Kelemework Gebreyohannes, Thomas Van Looy, Agnieszka Wozniak, Jasmien Wellens, Haifu Li, Jasmien Cornillie, Ulla Vanleeuw, Lise Vreys, Matthew Squires, Ana-Maria Rodringuez, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Anti-tumor effects of dovitinib, a multi-target kinase inhibitor, in patient-derived gastrointestinal stromal tumor (GIST) xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2015-775